Structural characterization of aminoglycoside 4′‐O‐adenylyltransferase ANT(4′)‐IIb from Pseudomonas aeruginosa

Abstract

Aminoglycosides were one of the first classes of broad‐spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4′)‐IIb in apo and tobramycin‐bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4′)‐IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4′)‐IIb structures revealed a two‐domain organization featuring a mixed β‐sheet and an α‐helical bundle. ANT(4′)‐IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4′)‐IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site‐directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4′)‐IIb‐represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.

AadA36, a novel chromosomal aminoglycoside nucleotidyltransferase from a clinical isolate of Providencia stuartii

“…ANT(3″) adenylates streptomycin and spectinomycin (but not tobramycin; Kehrenberg et al, 2005 ), while ANT(6) confers resistance to streptomycin ( Abril et al, 2010 ), and ANT(9) mediates resistance to spectinomycin ( Kanchugal and Selmer, 2020 ). ANT(4′) and ANT(2″), however, confer resistance to multiple antimicrobials, including tobramycin ( Wiedemann and Husing, 1985 ; Semper et al, 2020 ). Therefore, AadA36 could be distinguished from the other four subclasses of the ANT family and was assigned as a novel lineage of the ANT(3″)-Ia family.…”

1H,13C and15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution

“…The crystal structure of SARS-CoV-2 nsp1 folded domain (28)(29)(30) revealed the presence of an additional short -strand (aa 95-97), which is not found in the structure of SARS-CoV nsp1 determined by NMR (PDB: 2HSX). In our study of the full-length nsp1, the presence of -strand folded between aa 95 and 97 could not be confirmed.…”

“…However, the last 26-aa-long peptide in this C-terminal fragment has been shown to fold into two short -helixes upon binding to 40S ribosome subunit (7,8,17). The structure of folded N-terminal domain of SARS-CoV nsp1 has been determined by NMR (PDB:2HSX), and two Xray structures of the folded domain of SARS-CoV-2 nsp1 have been recently published (PDB:7K7P and 7K3N) (28)(29)(30). The N-terminal nsp1 domains of both viruses have similar folds.…”

The predictive potential of different molecular markers linked to amikacin susceptibility phenotypes in Pseudomonas aeruginosa

“…The approach implemented in this study has combined different measures of diagnostic accuracy in a complementary way in addition to functional evidence to investigate the candidate molecular markers. Although amikacin is known to be stable to the action of aminoglycoside inactivating enzymes, both aminoglycoside 6’-N-acetyltransferase Ib (AAC(6’)-Ib) [ 25 ] and aminoglycoside nucleotidyl transferase ( ant 4’-IIb) [ 26 ] have been reported to affect amikacin activity in Ps . aeruginosa .…”